Dopamine (DA) release in the cortex and basal ganglia is strongly implicated in modulation of CMS function and behavior and is thought to occur through a variety of potential secretory sites. Among these are axonal projections where small clear synaptic vesicles appear clustered in varicosities that resemble presynaptic terminals for typical fast-acting neurotransmitter secretion. Given that DA acts on much longer time scales than fast-acting neurotransmitters, the mechanism involved in controlling the presynaptic machinery may well be different than for those more typical "fast" synapses. Here we propose to examine details of the presynaptic vesicle cycle for these dopaminergic release sites. The long term objective of this proposal is to characterize the mechanism that control the presynaptic vesicle cycle for small clear dopaminergic veshicles. We will make use of technologies previously developed in the lab to examine many aspects of the molecular and biophysical nature of the presynaptic vesicle cycle in cortical and hippocampal cultures. These approaches rely heavily on optical techniques using exogenous organic probes such FM dye family members as well as genetically-encoded tags of presynaptic proteins that allow dynamic and quantitative information about the vesicle cycle to be obtained. These will be adapted to primary dissociated cell cultures of mid-brain neurons from the ventral tegmental area (VTA). We propose 3 specific aims to accomplish this initial characterization of the cell biological, physiological and biophysical aspects of the dopaminergic vesicle cycle. These include characterizing the properties of the vesicle pool in turns of depletion rates, replenishment rates, the sensitivity of pool turnover to stimulation at varied calcium concentrations, as well as the kinetics of endocytosis. Finally we will take advantage of the ability to detect dopamine sectretion directly using carbon-fiber amperometry to examine how details of the vesicle cycle impact neurotransmitter release.